Molecular cloning and characterization of the gene encoding N′-[(5′-phosphoribosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (BBM II) isomerase from Arabidopsis thaliana

We have isolated an Arabidopsis BBM II isomerase cDNA from an Arabidopsis cDNA library, by means of functional complementation of the E. coli hisA mutant strain HfrG6. The isolated cDNA encodes a polypeptide of 304 amino acids with a calculated molecular weight of 33?363. Sequence comparison with the HIS6 proteins of yeasts revealed that Arabidopsis BBM II isomerase contains an N-terminal extension of approximately 40 amino acids that shows the general properties of chloroplast transit peptides. This finding is consistent with the localization of other histidine biosynthetic enzymes, such as imidazoleglycerolphosphate dehydratase and histidinol dehydrogenase, in the chloroplasts in higher plants. The primary structure of the mature protein was 50% and 42% identical, respectively, to the HIS6 proteins of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, while no prominent sequence similarity to the bacterial BBM II isomerase was found. That the isolated Arabidopsis cDNA actually encodes a functionally active BBM II isomerase activity was confirmed in an in vitro enzyme assay using a crude extract prepared from strain HfrG6 transformed with the Arabidopsis BBM II isomerase cDNA.     

Fluorescent-labeled crosslinks in collagen: Pyrenebutyrylhydrazine

The aldehydes present in acid-soluble type I collagen react with pyrenebutyrylhydrazine to form various types of complexes under different reaction conditions. These complexes exhibit one or more of three different pyrene fluorescence bands: monomer, excimer, and aggregate fluorescence. Collagen, whose aldehydes have been reduced with NaBH₄, does not react with this fluorescent hydrazine, confirming that the hydrazine reacts specifically with aldehyde groups to form hydrazones. The absence of a reaction with pepsin-treated collagen also shows that the fluorescent labels are primarily in the nonhelical terminal telopeptides. Upon dialysis, the pyrene label bound to a saturated aldehyde in an α-chain is lost; whereas that bound to an unsaturated aldehyde remains on the protein. The pyrene monomer fluorescence in the β-chain of old collagen is stronger than that of young collagen. The formation of the pyrene excimer fluorescence implies the proximity of two pyrene molecules, probably attached to two adjacent aldehydes. Upon changing from acidic to neutral pH, both excimer and aggregate fluorescence bands disappear within a few seconds, revealing a very rapid alteration at the telopeptides.     

TSH-binding inhibitor immunoglobulin (TBII) in thyroidal venous blood and histological grade of intrathyroidal lymphocyte appearance in Graves' disease

The aim of this study is to investigate the possibility of determining the appropriate amount of thyroid tissue to leave behind as remnant weight in subtotal thyroidectomy for Graves' disease by measuring the TBII value, and correlating the outcome to the grade of intrathyroidal lymphocyte infiltration. We therefore have investigated the levels of TBII in the thyroidal (T-TBII) and peripheral (P-TBII) venous blood, and the grade of lymphocytic infiltration and lymph follicle formation in the intrathyroidal tissue. There was no parallel relationship between the T-TBII value and the grade of lymphocytic infiltration and lymph follicle formation. There was no marked difference between the T-TBII value and the P-TBII value. It was thus not possible to use these data to determine the proper remnant weight of Graves' thyroid tissue at the time of surgery.     

Temporal fluctuations of cytokine concentrations in human milk

Circadian rhythms affect the circulating levels of proteins that can modulate immune responses. Cytokines represent a group of proteins present in breast milk that modulate the immune system, but the effects of fluctuations of these proteins have not yet been elucidated. In this work, the cytokines present in human milk were quantified, taking into account the phases of the day and the maturation stages of human milk. Samples were collected at different stages of milk maturation and in two phases, day and night. The levels of cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, INF-γ and IL-17 were evaluated by flow cytometry. All quantified cytokines showed chronobiological variations with regard to the phase of day and/or stage of breast milk maturation. These data indicate that cytokines are subjected to fluctuations, and this knowledge is important for the use of human breast milk as an intervention strategy.     

Synthesis and biological evaluation of the natural product komaroviquinone and related compounds aiming at a potential therapeutic lead compound for high-risk multiple myeloma

Alternatives of treatments for multiple myeloma (MM) have become increasingly available with the advent of new drugs such as proteasome inhibitors, thalidomide derivatives, histone deacetylase inhibitors, and antibody drugs. However, high-risk MM cases that are refractory to novel drugs remain, and further optimization of chemotherapeutics is urgently needed.We had achieved asymmetric total synthesis of komaroviquinone, which is a natural product from the plant Dracocephalum komarovi. Similar to several leading antitumor agents that have been developed from natural compounds, we describe the antitumor activity and cytotoxicity of komaroviquinone and related compounds in bone marrow cells. Our data suggested that komaroviquinone-related agents have potential as starting compounds for anticancer drug development.